Single Cell Diagnosis of Leukemia using Multiplex Nested PCR System. To better direct targeted diagnostics and therapies to cancer, such as leukemia, there is a need for a simple blood-based assay that can provide expression information on a consistent basis with minimal patient discomfort. Current methods for chronic myelogenous (or myeloid) leukemia (CML) detection are based on the complete blood count, which shows increased granulocytes of all types. A bone marrow biopsy to screen for the Philadelphia chromosome is often performed as part of the evaluation for CML, but bone marrow morphology alone is insufficient to diagnose CML. In order to choose the best treatment and monitor the patient's response, it is important to know if the patient's type of leukemia is CML. To confirm the diagnosis, clinicians need to rely on a multiplex qRT-PCR assay using genes known to differentially express in express in different types of leukemia However, all current assay methods are laborious and time consuming, and no high throughput method exists for screening a large number of cells in blood samples. Therefore, Maxwell Sensors Inc. proposes to develop a Single Cell Diagnosis of Leukemia using the Nanodroplet based Multiplex Nested PCR (NMNP) System, which has the capacity to process thousand of individual cells;detect circulating tumor cells based on multiplexed PCR results;and analyze the presence or absence of chimeric BCR/ABL mRNAs for chronic myeloid leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). The NMNP system automates all the sampling processes, preparing and detecting low abundance genes in single cells that are often lost in the extraction process. The main advantages of the proposed NMNP system are 1) high throughput: processing thousands (~50,000) of single cells simultaneously, 2) single cell analysis: nucleic acid detection at the single cell level, 3) multiplex PCR: carries out nested PCR for several genes in single circulating tumor cells, and 4) automated system: single cell manipulation and detection of the PCR results on a chip. The focus of the Phase I work is to design and fabricate a high-throughput nanodrop chip, integrate the NMNP Chip with fluidics and an optical detection system, develop the NMNP multiplex PCR protocol and bioassay, characterize the chip, and evaluate NMNP performance. PUBLIC HEALTH RELEVANCE: Maxwell Sensors Inc. proposes to develop a two step Multiplex Nested PCR system using single circulating tumor cells for cancer diagnosis. This system combines nanotechnology, fluid dynamics, real-time Multiplex PCR, single-cell assays, and circulating tumor cell cancer diagnostics. These assays also have the potential to guide treatment selection based on the molecular profile of the tumor before therapy. Detection of abnormal genes in the intracellular materials provides important clues for early disease diagnosis.